Attenuators

Abstract

Methods for detecting nucleic acid sequences, where attenuator oligonucleotides are provided to reduce the number of detection products resulting from highly abundant sequences.

We claim: 
     
       1. A set of attenuator oligonucleotides for a sample type that has a plurality of relatively high abundance target sequences (HATs), each HAT having a downstream region (DR) and an adjacent upstream region (UR),
 wherein the set comprises for each HAT: 
 (a) a downstream detector oligo (DDO) comprising a P 1  primer sequence and a region complementary to the downstream region of the HAT target sequence (DR′), and 
 (b) an upstream detector oligo (UDO) comprising a region complementary to the upstream region of the HAT target sequence (UR′) and a P 2 ′ primer hybridization sequence,
 wherein both the downstream detector and the upstream detector are capable of hybridizing simultaneously to the HAT target sequence under predetermined hybridization conditions, and; 
 
 (c) an attenuator oligo comprising a portion of the DR′ or the UR′ of sufficient length to specifically bind to a corresponding portion of the DR or UR of the HAT target sequence,
 and further comprising a sequence X that is different from P 1  or a sequence Y that is different from P 2 ′. 
 
 
     
     
       2. The set of attenuator oligonucleotides of  claim 1 , further comprising a pool of primer oligos comprising a P 1  sequence and a pool of primer oligos comprising a P 2  sequence. 
     
     
       3. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator comprises a portion at least partially complementary to one of the DR or UR of a HAT, and further comprises a portion at least partially complementary to the other region of the HAT. 
     
     
       4. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator comprises a portion that is complementary to at least a portion of the DR of a HAT and another portion that is complementary to at least a portion of the UR of the HAT. 
     
     
       5. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator comprises at least one mismatched base relative to the DR or UR of a HAT. 
     
     
       6. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator comprises a portion partially complementary to the DR of a HAT, and comprises 1, 2, or 3 mismatched bases at any of the positions at the 3′ end or at 1 or 2 positions from the 3′ end. 
     
     
       7. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator comprises a portion partially complementary to the UR of a HAT, and comprises 1, 2, or 3 mismatched bases at any of the positions at the 5′ end or at 1 or 2 positions from the 5′ end. 
     
     
       8. The set of attenuator oligonucleotides of  claim 1 , wherein, under the hybridization conditions in (b), the sequence X does not specifically bind to the P 1  sequence or its complement; or the sequence Y does not specifically bind to the P 2  sequence or its complement. 
     
     
       9. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator comprises a nonamplifiable segment comprising a reverse complementary sequence. 
     
     
       10. The set of attenuator oligonucleotides of  claim 1 , wherein the DR of a HAT is connected to the UR of the HAT by a flexible linker. 
     
     
       11. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator is phosphorylated. 
     
     
       12. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator is not phosphorylated. 
     
     
       13. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator further comprises a portion that is complementary to a region adjacent to the DR or UR of a HAT. 
     
     
       14. The set of attenuator oligonucleotides of  claim 1 , wherein the DR and UR of a HAT are separated by one or more nucleotides, and an attenuator has a portion that is complementary to the one or more nucleotides. 
     
     
       15. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator comprises a selectively cleavable site. 
     
     
       16. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator further comprises a nonextendable or nonligatable blocking group. 
     
     
       17. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator is detectably labeled. 
     
     
       18. The set of attenuator oligonucleotides of  claim 1 , wherein an attenuator comprises a barcode sequence. 
     
     
       19. The set of attenuator oligonucleotides of  claim 18 , wherein the barcode sequence is specific to a HAT. 
     
     
       20. The set of attenuator oligonucleotides of  claim 1 , wherein a HAT comprises a sequence partially encoding a product selected from the group consisting of cytochrome c, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L7 (RPL7), ribosomal protein S6 (rpS6), snRNA RNUs, phosphoglycerokinase (PGK), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), beta-actin, and beta-tubulin. 
     
     
       21. The set of attenuator oligonucleotides of  claim 1 , wherein the attenuator comprises a portion of the DR′ and a portion of the UR′. 
     
     
       22. The set of attenuator oligonucleotides of  claim 9 , wherein the reverse complementary sequence forms a hairpin loop under the hybridization conditions of (b).